Metabolic rewiring overcomes physiological constraints in Sphingobium lignivorans SYK-6 for valorization of industrial lignin streams.

Lignin-rich industrial streams represent an abundant but underutilized source of renewable aromatic carbon. Efficient biological conversion requires microbial hosts capable of metabolizing chemically diverse lignin-derived aromatic compounds; however, such capabilities are typically found in environmental bacteria, which are constrained by physiological and metabolic limitations. Sphingobium lignivorans SYK-6 harbors extensive aromatic catabolic pathways, but its inability to utilize glucose and its methionine auxotrophy have limited its use as a production host. Here, we identified the metabolic basis of these constraints and systematically rewired the underlying pathways to overcome them. Introduction of a heterologous glucose transporter, reconstruction of methionine biosynthesis, chromosomal integration of pathway genes, and adaptive laboratory evolution collectively enabled robust growth on glucose while eliminating methionine auxotrophy. The engineered strain converted lignin-derived aromatics in oxygen-soda-anthraquinone pulping black liquor derived from Japanese cedar, achieving high-yield production of the polymer precursor 2-pyrone-4,6-dicarboxylic acid (PDC) (2.71 g/L and >130 mol% conversion relative to major quantified aromatics). We further show that gluconolactonase can substitute for 6-phosphogluconolactonase in the Entner-Doudoroff pathway, demonstrating that central carbon metabolism can accommodate functionally analogous enzymes. Together, these results provide a metabolically rewired SYK-6 strain as a platform for the valorization of industrial lignin streams and suggest that overcoming physiological and metabolic constraints can enable non-model aromatic-degrading bacteria to function as industrial production hosts.